The present invention relates to compounds and methods for elevating endogenous insulin-like growth factors and their activities in living bodies.
Insulin-like growth factors (hereinafter xe2x80x9cIGFxe2x80x9d) are found in two distinct molecular forms called IGF-I and IGF-II, respectively. Human IGF-I and IGF-II are 70 and 67 amino acids in length, respectively. Compared to IGF-II, IGF-I has three more amino acids at the site corresponding to the C peptide, which is a partial structure of insulin. The amino acid sequence homology between IGF-I and IGF-II is about 60% while that between IGF-I and insulin is about 40%. Although the liver and kidney are the major sites of production for IGF-I in living bodies, northern blot analysis of MRNA has revealed that IGF-I is produced by almost all tissues in the body (D""Ercole, A. J., et al., Proc.
Natl. Acad. Sci. USA. 81, 935 (1984); Humbel, R. E., et al., Eur. J. Biochem., 190, 445 (1990)). IGF-I is considered to act not only as an endocrine factor but also as paracrine or autocrine factor.
IGF-I and IGF-II bind to distinct and specific receptors; an IGF-I receptor and an IGF-II/cation-independent mannose-6-phosphate receptor, respectively. However, since IGF-II has also been shown to bind to the IGF-I receptor, the various biological activities associated with IGF-II are thought to occur mainly through the IGF-I receptor located on cell surface (Casella, S. J., et al., J. Biol. Chem., 261, 9268 (1986); Sakano, K., et al., J. Biol. Chem., 266, 20626 (1991)).
The IGF-I receptor shares a high degree of amino acid sequence homology with the insulin receptor and the two molecules resemble each other in their intracellular signal transduction mechanism (Shemer, J., et al., J. Biol. Chem., 262, 15476 (1987); Myers, M. G. Jr., et al., Endocrinology, 132,. 1421 (1993)). IGFs regulate glucose metabolism predominantly in the peripheral tissue, which is different from insulin, as shown in animal model studies. The receptors for IGFs and insulin are differentially localized in tissues, and this may explain why the biological effect of IGFs in the body is distinguishable from insulin""s effect (Laager, R., et al., J. Clin. Invest., 92, 1903 (1993)).
The blood of an average adult human contains about 100 nM of IGF and about 100 pM of insulin (Baxter, R. C., in Modern Concepts of Insulin-Like Growth Factors (Spencer, E. M., ed) pp.371, Elsevier Science Publishing Co., New York-Amsterdam (1991)). Most IGFs found in living bodies form complexes with an IGF-binding protein (hereinafter xe2x80x9cIGFBPxe2x80x9d). It appears that a specific binding protein exists for each IGF. The hypoglycemic effect of free IGF or unbound IGF is about 5 to 10% of that of insulin (Guler, H. P. et al., New Engl. J. Med., 317, 137 (1987)), indicating that insulin-like growth factors are at concentrations of about 50 to 100-fold greater than insulin (Baxter, R. C., in Modern Concepts of Insulin-Like Growth Factors (Spencer, E. M., ed) pp.371, Elsevier Science Publishing Co., New York-Amsterdam (1991)).
The World Health Organization has classified the disease, Diabetes mellitus, into roughly three categories on the basis of their distinct clinical patterns:
(1) Insulin-dependent diabetes mellitus (hereinafter xe2x80x9cIDDMxe2x80x9d)
(2) Non insulin dependent diabetes mellitus (hereinafter xe2x80x9cNIDDMxe2x80x9d)
(3) Other diabetes mellitus (derived from pancreato-pathy diseases or endocrinopathy)
A method for treating IDDM involves insulin therapy, while diet therapy, kinesitherapy, or treatment with an oral hypoglycemic agent or with insulin is mainly used in the treatment of NIDDM. In recent years, IGF-I therapy has been considered as an alternative treatment for insulin-dependent diabetes mellitus in cases where administration of insulin alone is not effective (Kuzuya, H., et al. Diabetes 42, 696 (1993)). Also for NIDDM, effects of IGF have been under investigation (Zenobi, P. D., et al., J. Clin. Invest., .90,. 2234 (1992); Moses, A. C., et al. , Diabetes, 45, 91(1996)).
Guler et al. observed that the intravenous injection of IGF-I into adult humans in an amount of 100 xcexcg/kg resulted in the lowering of blood glucose levels with the lowest level occurring after 20 minutes (Guler, H. P., et al., New Engl. J. Med., 317, 137 (1987))
Takano et al. observed that hypoglycemic activity was observed in adult humans following the subcutaneous injection of IGF-I in an amount of 60 to 120 xcexcg/kg, and that administration of IGF-I every 6 days in an amount of 100 xcexcg/kg lowered the uric acid and creatinine levels in blood (Takano, K., et al., Endocrinol. Jpn., 37, 309. (1990)).
In addition, there are reports on the lowering of free fatty acid levels in blood (Turkalj. I., et al., J. Clin. Endocrinol. Metab., 75, 1186 (1992)), the lowering of neutral fats such as triglyceride (Turkalj. I., et al., J. Clin. Endocrinol. Metab. 75, 1186 (1992); Zenobi, P. D. , et al. , J. Clin. Invest., 90, 2234 (1992)), and the lowering in total cholesterol level (Zenobi, P. D., et al., Diabetologia 36, 465 (1993)). Increases in renal blood flow and glomerular filtration rate (Elahi, D., et al., in Modern Concepts of Insulin-Like Growth Factors (Spencer, E. M., ed) pp2l9, Elsevier Science Publishing Co., New York-Amsterdam (1991)), have been reported for IGF-I.
There is also a report that the administration of IGF-II was effective for intractable diabetes mellitus (Usara, A., et al., Diabetes, 44, Suppl. 1, 33A, 1995)). Results from animal model studies suggest the effectiveness of IGF-I in the reduction of conditions associated with stress including glucose metabolism at the time of hemorrhagic shock, the alleviation of side effects caused by sugar infusion (Unexamined Japanese Patent Publication (KOKAI) No. Hei 7-242565).
Administering IGF to animals has helped to identify the numerous biological activities of IGF including hypoglycemic activity, induction of proliferation, cell differentiation, and anobolic activity. Local administration of IGF-I to the injured peripheral nervous system results in the proliferation of non-neural cells while stimulating neurons. It is reported that IGF-I receptors are present on spinal cells and that administration of IGF-I decreases cell death of motor neurons. In addition, it is recognized that the administration of IGF increases the muscular end plate, promotes the functional recovery of a damaged sciatic nerve and prevents peripheral motor paralysis observed during chemotherapy (Sjoberg, J., et al., Brain Res. 485, 102 (1989).
Based on these foregoing experimental observations in the peripheral nervous sytem, clinical tests using IGF-I in the treatment of amyotrophic lateral sclerosis and degenerative diseases of the motor neuron have been conducted (Lewis, M. E., et al., Exp. Neurol., 124, 73(1993)). Similarly, the use of IGF in promoting the survival of neuronal cells is recognized as being important in the treatment of Alzheimer""s disease, apoplexy, amyotrophic lateral sclerosis, Parkinson""s disease and the like (Unexamined Japanese Patent Publication (KOHYO) No. Hei 6-510305). In addition, the effectiveness of IGF-I in the treatment of muscular dystrophy has also been reported (Vlachopapadopoulou, E., et al., J. Clin. Endocrinol. Metab., 80, 3715 (1995)).
The effects of IGF on diabetic neuropathy have also been studied. In an IDDM rat model (STZ-rat: streptozotocin-diabetic rat model), alleviation of diabetic neuropathy was observed when IGF was administered at concentrations that did not lower blood glucose levels (Zhuang, H-X, et al., Exp. Neurol., 140, pp198-205 (1996)). It has also been reported that administration of IGF in an NIDDM rat model (diabetic obese Zucker (fa/fa) rat), reduced the level of IGF-II mRNA in the sciatic nerve, spinal nerves and brain nerves, and alleviated the diabetic neuropathy when used at a concentration that did not result in lowering of blood glucose levels (Zhuang, H-X, et al., J. Pharmacol. Exp. Ther., 283, pp366-374 (1997)). These findings suggest that IGF is effective in the treatment of diabetic neuropathy.
The effects of IGF have also been studied on cardiac function. When doxorubicin is administered to rats, it causes myocardiopathy, but administration of IGF-I improves myocardial function (Ambler, G. R., et al., Cardiovasc. Res., 27, 1368 (1993)). Consequently, IGF-I is thought to be useful in the prevention and treatment of myocardiopathies including myocarditis and myocardial infarction, cardiac disease, and acute attack via its ability to increase cardiac rate and improve cardiac output (Unexamined Japanese Patent Publication (KOHYO) No. Hei 6-504286).
The effects of IGF-I on acute renal insufficiency caused by ischemia have also been reported. On day 5 after an ischemic attack, IGF-I was administered three times daily by subcutaneous injection for three days. The result was that IGF improved renal function, promoted formation of new renal tubules, inhibited proteolysis and promoted protein synthesis, and decreased catabolism (Ding, H., et al., J. Clin. Invest., 91, 2281 (1993)).
It has also reported that the local administration of IGF-I to a skin injury, i.e., wounds, burn injuries or the like, reduces the length of recovery. In a burn injury model, the administration of rat IGF-I increased body weight, weight of the enteromucosa, mucosal DNA and protein expression, and decreased the transfer of enterobacterium to the intestinal lymph gland, thereby improving intestinal function and life prognosis (Huang, K. F., et al., Arch. Surg., 128, 47 (1993)).
Together with a platelet-derived growth factor (hereinafter xe2x80x9cPDGFxe2x80x9d), IGF-I promotes mitosis and protein synthesis of cultured mesenchymal cells, and although curing of skin disorders is not promoted by the single use of PDGF or IGF-I, the combined use of both factors promotes the growth of connective and epithelial tissues (Stiles, C. D., et al., Proc. Natl. Acad. Sci. USA, 76, 1279 (1987)). In another report, however, the single application of either one of these growth factors was shown to stimulate wound healing (Tsuboi, R., et al., J. Exp. Med., 172, 245 (1990)). Therefore, attempts have been made to use IGF for promoting wound healing (Unexamined Japanese Patent Publication (KOKAI) No. Sho 63-233925, Unexamined Japanese Patent Publication (KOHYO) Nos. Hei 3-505870 and Hei 6-506191, and Unexamined Japanese Patent Publication (KOKAI) No. Hei 7-316066).
In addition, IGF-I is effective at improving immune function. IGF-I is produced in the thymus and sites of inflammation and is considered to be important in the regulation of proliferation and function for T lymphocytes expressing the IGF-I receptor (Tapson, V. F., et al., J. Clin. Invest., 82, 950 (1988)). It is reported that IGF-I promotes the proliferation of lymphocytes at nanomolar concentrations (Schimpff, R. M., et al., Acta Endocrinol. 102, 21 (1983)). Accordingly, the use of IGF-I for treatment of immunodeficient patients including AIDS patients is under investigation (Unexamined Japanese Patent Publication (KOHYO) No. Hei 6-508830).
Moreover, IGF-I is considered to be effective in the treatment of osteoporosis since increases in bone mass have been associated with IGF (Bennett, A. E., et al., J. Clin. Endocrinol. Metab., 59, 701 (1984); Brixen, K., et al., J. Bone. Miner. Res., 5, 609 (1990); Johannsson, A. G., et al., J. Intern. Med., 234, 553 (1993); Johannsson, A. G., et al., Lancet, 339, 1619 (1992); Riggs, B. L., Am. J. Med., 95, Suppl.5A, 62S, (1993); Unexamined Japanese Patent Publication (KOKAI) No. Hei 4-235135 and U.S. Pat. No. 4,861,757).
However, it is recognized that in their naturally occurring state, almost all of the IGFs form complexes with IGFBP in living bodies, which effectively serves to regulate their physiological activity in vivo (Rechler, M. M., Vitam. Horm., 47, 1 (1993); Clemmons, D. R., Growth Regul. 2, 80 (1992)).
Six IGFBPs (designated xe2x80x9cIGFBP-1 to IGFBP-6xe2x80x9d) have been identified thus far, and each of them exhibits a high degree of amino acid sequence identity or homology. The homology is markedly similar in the N-terminal and C-terminal regions where many of the cysteine residues are located, while the proteins are much less homologous in their intermediate domains. For all six human IGFBPs, the respective positions for sixteen (16) cysteine residues is conserved (IGFBP-1 to IGFBP-5 have conserved 18 cysteine residues) (Shimasaki, S., et al., Prog. Growth Factor Res., 3, 243 (1991)).
The concentrations of IGFBP-1, IGFBP-2 and IGFBP-3 in the blood of an adult human are about 2 nM, 5 nM and 100 nM, respectively, and IGFBP-3 is a major binding protein for IGF (Baxter, R. C., in Modern Concepts of Insulin-Like Growth Factors (Spencer, E. M., ed) pp371, Elsevier Science Publishing Co., New York-Amsterdam (1991)). When normal human serum is fractionated by gel filtration under neutral conditions, the IGFs elute in the vicinity of 150 kDa and are found as a ternary complex (Baxter, R. C., et al., Proc. Natl. Acad. Sci. USA, 86, 6898 (1989)). This ternary complex is composed of IGF-I (or IGF-II) (m.w. of about 7.5 Kda), IGFBP-3 (m.w. of 53 Kda and inert to acid) and a subunit protein (m.w. of 84 KDa and labile to acid (Acid Labile Subunit or xe2x80x9cxcex1-subunitxe2x80x9d; hereinafter xe2x80x9cALSxe2x80x9d)). It is hypothesized, that when IGF binds to IGFBP-3, the major binding protein in blood, ALS binds to this binary complex to form a ternary complex having a total m.w. of 150 KDa.
It is considered that free IGF or a binary complex of IGF and IGFBP are able to pass through the capillary wall, while the ternary complex cannot (Rechler, M. M., Vitam. Horm., 47, 1(1993)). As regards the half-life of human IGF in blood, that of free IGF is as short as about 10 minutes, that of the binary complex of IGF and IGFBP is about 30 minutes and that of the ternary complex composed of IGF, IGFBP-3 and ALS is about 15 hours (Zapf, J., et al., in Modern Concepts of Insulin-Like Growth Factors (Spencer, E. M., et) pp.591, Elsevier Science Publishing Co., New York-Amsterdam (1991)).
As a result of the ternary complex formation, the half-life of IGF in blood is extended and its physiological activity is suppressed. Also, formation of a binary complex of IGF and IGFBP, extends the half-life of IGF in blood and is involved in the regulation of the physiological activity of IGF (Baxter, R. C., et al., Prog. Growth Factor Res., 1, 49 (1989)).
Little or no difference is observed in ALS among species, and homology of ALS between a human and a rat is 78% (Dai, J., et al., Biochem. Biophys. Res. Commun., 188, 304 (1992)). Earlier, it was reported that ALS alone does not bind to IGF or IGFBP-3, but more recently ALS has been shown to exist as a complex with IGFBP-3 in the serum of rats (Lee, C. Y., Endocrinology, 136, 4982 (1995)).
IGF administration to a living body does not elevate the concentration of free IGF in blood, but elevates the concentration of IGFBP-2. It is now considered that an IGF-dependent mechanism exists in the living body for regulating the expression of IGFBP (Zapf, J., et al., in Modern Concepts of Insulin-Like Growth Factors (Spencer, E. M., et) pp.591, Elsevier Science Publishing Co., New York-Amsterdam (1991)).
One regulatory mechanism associated with the ternary complex of IGF, IGFBP-3 and ALS is seen with non-islet cell tumor hyperglycaemia (hereinafter xe2x80x9cNICTHxe2x80x9d). Non-islet cell tumors, which produce IGF-II, are associated with hypoglycaemia or low blood glucose levels. NICTH-derived IGF-II is glycosylated in the E-domain of the precursor protein. Normally, IGF-II exists as a 7.5 kDa nonglycosylated protein.
Glycosylated IGF-II forms a complex with IGFBP-3, but cannot form a ternary complex with IGFBP-3 and ALS (Baxter, R. C., in Modern Concepts of Insulin-Like Growth Factors (Spencer, E. M., ed) pp371, Elsevier Science Publishing Co., New York-Amsterdam (1991)) so the glycolsylated form is considered to exert blood glucose lowering action because of its inability to form complexes with ALS. Glycosylated IGF-II is complexed with IGFBP-3 in blood, and this complex is thought to pass through the capillary vessel wall (Rechler, M. M., Vitam. Horm., 47, 1 (1993)) and to reach the target site. Administration of the IGF-I and IGFBP-3 complex to hypophysectomized rats, demonstrated IGF-I activity, although weaker than the administration of IGF-I alone (Zapf, J., et al., J. Clin. Invest., 95, 179 (1995)). On the basis of these results, the complex of IGF and IGFBP-3 found in blood, is presumed to be transported to target tissues or organs where the activity of IGF is demonstrated.
The IGF-IGFBP complex can also be regulated by a IGFBP-specific protease. IGFBP-3 concentrations in the blood of a gravida during the last stage of pregnancy are slightly increased when measured by a radioimmunoassy (hereinafter xe2x80x9cRIAxe2x80x9d) using an anti-IGFBP-3 antibody. However, when blood samples from the same patient are analyzed by a western blot method using 125I-IGF, a decrease in IGFBP-3 concentration is observed. This apparent discrepency in results is explained by the presence of a protease found in the blood of the gravida, which specifically degrades IGFBP-3 (Hossenlopp, P., et al., J. Clin. Endocrinol. Metab., 71, 797 (1990); Giudice, L.C., et al., J. Clin. Endocrinol. Metab., 71, 806 (1990)). As a result of this proteolytic activity on the IGFBP-3 protein, the affinity between IGF and IGFBP-3 is lowered, and a direct increase in IGF activity is observed.
These observations demonstrate that activity of IGF in the living body is highly regulated by IGFBP and ALS. Exogenous IGF when administered is rapidly metabolized or complexed with IGFBP or ALS. Even if IGF or a compound having IGF-like activity is administered exogenously, the IGF activity is controlled by IGFBP and/or ALS in the living body.
The inventors have found that endogenous IGF can be increased by administering compounds which release IGF from binary (IGF-IGFBP) and ternary (IGF-IGFBP-ALS) complexes or which increase binary complex formation having IGF-like activity.
The compounds of the present invention have at least one the properties of: converting a binary IGF-IGFBP complex or a ternary IGF-IGFBP-ALS complex into free IGF; converting the ternary complex into the binary IGF-IGFBP complex; dissociating the ternary complex into IGF or the binary IGF-IGFBP complex; or inhibiting the formation of the binary IGF-IGFBP complex or the ternary IGF-IGFBP-ALS complex.
An object of the present invention is to utilize the abundant amount of endogenous IGF which is otherwise physiologically regulated by complexing with IGFBP and or ALS.
Another object of the invention is a method for elevating the concentration of IGF, comprising converting complexed IGF into free IGF.
Another object of the present invention is a method for elevating the concentration of the binary complex, which has lower IGF activity than free IGF but higher IGF activity than the ternary complex.
Biologically active, unbound IGF can be obtained by:
conversion of the IGF-IGFBP complex or IGF-IGFBP-ALS complex into IGF;
dissociation of IGF from the IGF-IGFBP complex or IGF-IGFBP-ALS complex; or
inhibition of the binding of IGF and IGFBP or binding of IGF, IGFBP and ALS.
A biologically active complex of IGF-IGFBP can be obtained by:
conversion of the ternary complex to the binary complex;
dissociation of the binary complex from the ternary complex; or
inhibition of the binding of the binary complex to ALS.
The concentrations of free IGF or the binary complex can be increased by:
1) a compound which coverts the binary complex in the living body into free IGF;
2) a compound which dissociates free IGF from the binary complex in the living body;
3) a compound which inhibits the binding of IGF and IGFBP in the living body;
4) a compound which converts the ternary complex in the living body into the binary complex;
5) a compound which dissociates the binary complex from the ternary complex in the living body;
6) a compound which inhibits the binding of the binary complex in the living body to ALS;
7) a compound which converts the ternary complex in the living body into free IGF;
8) a compound which dissociates [dissociates] IGF from the ternary complex in the living body;
9) a compound which inhibits the binding of IGF, IGFBP and ALS in the living body;
10) a compound which binds to IGFBP but does not bind to an IGF receptor or an insulin receptor;
11) an IGF derivative which binds to IGFBP but does not bind to an IGF receptor or an insulin receptor;
12) an IGF derivative having the addition, deletion or substitution of one or more amino acid residues, and which binds to IGFBP but does not bind to an IGF receptor or an insulin receptor;
13) an IGF derivative having an amino acid sequence similar to human IGF-II except that the tyrosine residue at amino acid position 27 and the valine residue at amino acid position 43 have been substituted with a leucine residue, and which binds to IGFBP but does not bind to an IGF receptor or an insulin receptor;
14) an anti-IGFBP antibody which binds to IGFBP but does not bind to an IGF receptor or an insulin receptor;
15) an anti-IGFBP antibody which binds to IGFBP-3 but does not bind to an IGF-I receptor or an insulin receptor.
Another aspect of the present invention is a medicament comprising a compound which can elevate the concentration of free IGF or the binary complex.
Another aspect of the present invention is a screening method for identifying a compound which can elevate the concentration of free IGF or the binary complex.